Nitric oxide (NO) has been shown to modulate biological processes in diverse cellular systems. In part the multifaceted biological functions of NO are facilitated through the modification of protein cysteine residues to S-nitrosocysteine. Until recently our ability to interrogate complex biological systems for these novel protein targets of NO has been restricted by the lack of complementary, validated proteomic methodologies. Although the development of the biotin-switch approach has in part advanced the discovery of protein targets, concerns with validation of the protein targets and issues with sensitivity limit the application of this methodology. Therefore, we have developed and propose to further refine a methodology that can specifically identify the S-nitrosocysteine residues in complex biological samples. The method employs selective peptide capturing and site-specific adduct mapping by liquid chromatography-tandem mass spectrometry. In human aortic smooth muscle cells, starting with a total of 4 nmoles protein S- nitrosocysteine per mg of protein this strategy identified 18 S-nitrosocysteine-containing peptides belonging to 16 proteins. In this application we propose to develop complementary approaches for direct labeling and capture of the S-nitrosocysteine adducts. Moreover, using isotopic labeling of cellular proteins or of S- nitrosocysteine adducts we propose to develop and implement a quantitative method for the S- nitrosocysteine proteome. Combining the power of selective S-nitrosocysteine peptide identification with quantitative proteomics will provide for the first time a global evaluation of the dynamic changes in levels of specific S-nitrosoproteins in living cells. These methodologies in conjunction with immunohistochemical and high resolution immuno-electron microscopy will be applied to define the temporal and spatial changes of three protein targets of S-nitrosylation, vinculin, 14-3-3theta and mitochondrial heat shock protein 70, which regulate critical aspects of vascular smooth muscle biology. Overall the proposed global and targeted proteomic approaches will explore the biological significance of S-nitrosocysteine in signaling pathways and protein networks in the cardiovascular system. Moreover the global characterization of the S-nitrosocysteine proteome will significantly advance our understanding of the biological functions of NO by uncovering previously unrecognized molecular targets for this molecule.